Cellular signaling by double-stranded RNA will be investigated in this project. DsRNA is a potent regulator of enzyme activation and transcriptional signaling. Its cellular actions are connected to the functioning of the interferon system and often it is a mediator of the cellular responses to virus infection. Its identity as a marker of viral infection has been reinforced by the recent discovery of its receptor, the Toll-like receptor 3 (TLR3), which is a component of the innate immune system that responds to various infectious agents. Additional interests in the biological actions of dsRNA have risen from the increasing usage of siRNA, which are short dsRNAs, as an experimental tool. The overall objective of our project is to identify the repertoire of human genes induced by activation of the innate immune system by dsRNA and viruses and to delineate the corresponding signaling pathways using biochemical and genetic tools. Specifically, we will analyze the convergent and divergent pathways used by dsRNA, Sendai virus and interferons to induce transcription of overlapping sets of genes For this purpose, we will use microarray analyses of genes induced by the above agents in various mutant cell lines defective in known components of relevant signaling pathways. We will determine the biochemical basis of the newly observed need of tyrosine phosphorylation of TLR3 in mediating signals generated by dsRNA. We will also investigate the biochemical, cellular and physiological functions of PACT, the protein activator of the multi-functional protein kinase, PKR, an intracellular receptor of dsRNA. A possible role of PACT in mediating innate immune responses through TLRs will also be investigated. Successful completion of the proposed project will lead to a better understanding of the mechanism of action of the mammalian innate immune system and how it regulates host-virus interactions and cell growth.